Prevalence of Acinetobacter baumannii bacteremia in intensive care units of Ibn Rochd University Hospital, Casablanca

Background and Objectives: Acinetobacter baumannii bacteremia are grave because of the multi-resistance of the organism to antibiotics. This study aimed to determine the prevalence of A. baumannii isolated from blood cultures and to describe their antibiotic resistance patterns. Materials and Methods: A retrospective longitudinal study was conducted on blood cultures between 2010 and 2014 from all Ibn Rochd University Hospital intensive care units; it was based on the exploitation of microbiology laboratory database (duplicates were excluded). Isolation and identification of A. baumannii were performed according to standard techniques of bacteriology and susceptibility testing as recommended by the CLSI. PCR was used to detect β-Lactamase genes, blaOXA-51, blaOXA-23. Results: Among the 4232 samples received at the laboratory, 2402 (56.8%) were positive. Negative coagulase Staphylococcus was isolated in 21.6% of cases followed by A. baumannii (9.2%), and K. pneumoniae (9.1%). A. baumannii strains were resistant to most antibiotics tested: imipenem (75.7%), ceftazidim (85.4%), cefotaxim (98.6%), gentamicin (78.1%), amikacin (63.5%) and ciprofloxacin (88.2%). All A. baumannii strains, resistant to carbapenem, tested were positive for blaOXA-51 genes and 87.5% expressed the blaOXA-23 genes. Conclusion: A. baumannii was the second germ frequently isolated from blood cultures in intensive care units. It was multi-resistant to antibiotics. The strengthening of hospital hygiene measures and surveillance of antibiotic resistance is needed to limit the spread of germs and to optimize the management of antibiotics.


INTRODUCTION
Acinetobacter baumannii is a Gram-negative pathogen. It is frequently associated with nosocomial infections (bacteremia, pneumonia, meningitis and urinary tract infections). It was also recognized worldwide as an emerging cause of nosocomial outbreaks and listed by the American Society of Infectious Diseases (IDSA) as one of the six most hazardous microorganisms (1). A. baumannii bacteremia in intensive care units (ICUs) are responsible of a significant morbidity and mortality and create a therapeutic problem due to the multidrug resistance of the organism to antibiotics (2,3). A. baumannii have a natural resistance to antibiotics, but also an acquired resistance by production of enzymes: the β-lactamases and especially metallo-β-lactamase and oxacillinases and by efflux pumps and changing porins (4). Carbapenem hydrolyzing β-lactamases belonging to oxacilinases enzymes are the main enzymes contributing to the inactivation of imipenem in A. baumannii (5,6).
The aim of this study was to determine the prevalence of A. baumannii isolated from blood cultures performed in intensive care units of Ibn Rochd University hospital at Casablanca-Morocco during the last five years (2010-2014) and to describe the evolution of the organism's resistance to antibiotics. Then, to detect the presence of Oxacillinases: OXA-23 and OXA-51 among the isolates showing resistance to imipenem.

Samples collection (Patients samples).
A retrospective longitudinal study was carried out on blood cultures from all ICUs in Ibn Rochd University Hospital between 2010 and 2014. It was based on the exploitation of the Microbiology laboratory database (duplicates of the same patient were excluded). All bacteria including A. baumannii identified by the standard bacteriological methods in the routine diagnosis of bacteremia from all ICUs were examined. PCR was used to target the bla OXA-23 and bla OXA-51 among the imipenem resistant isolates A. baumannii.
Preparation of DNA and PCR. All A. baumannii resistant to imipenem were grown on Mueller-Hinton (MH) agar plates (Bio-Rad, Marnes-la-Coquette, France) for 18-24 hours at 37°C, bacterial cells were suspended in 500 μl of ultrapure water. Suspension was heated at 100°C for 10 min and immediately frozen at 0°C for 5 min. 300 μl of supernatant were then recovered after centrifugation of 14000 g for 10 min. Supernatant containing DNA was stored at -20°C until further use (5).
PCR protocol described by Sandle et al. (6) was used for detection of bla OXA-23 and bla OXA-51 among the imipenem resisrant isolates .
Statistical analysis. Data were analyzed with Epi-Info 7 (Centers for Disease Control, Atlanta, Georgia, USA) and Microsoft Excel. The chi square test or Fisher's exact test was performed to compare proportions. Differences were considered significant if the p-value is <0.05.

RESULTS
During the study period, a total of 4232 non-duplicate blood cultures from 4232 patients were received at the laboratory. Of these, 2402 blood cultures (56.8%) were positive. Negative coagulase Staphylococcus was isolated in 21.6% of cases followed by A. baumannii (9.2%) and K. pneumoniae (9.1%) (Fig. 1).

DISCUSSION
In our study, A. baumannii was the second germ frequently isolated from blood cultures in ICUs between 2010 and 2014 (9.2%). It was characterized by a multi-resistance to the antibiotics tested (C3G, aminoglycosides, fluoroquinonolones and imipenem). It was however, sensitive to colistin in 99% of cases.
The evolution of antibiotic resistance during the study period was increasing especially for imipenem (from 50% in 2010 to 75% in 2014). All isolates of A. baumannii, resistant to carbapenem, were positive for bla OXA-51 genes and 87.5% were positive for bla  .
A. baumannii is considered as an opportunistic pathogen that can survive in austere conditions. It is responsible of an increasing rate of severe nosocomial infections. They affect especially immunocompromised patients, exposed to prolonged stays in ICUs and having a previous exposure to antibiotics; carbapenems and 3 rd generation cephalosporins are the most involved, followed by fluoroquinolones, aminoglycosides and metronidazole (2,7). Other factors that are  The distribution of bacteria in our study is consistent with a previous study in the same hospital in which A. baumannii represented 13% of the bacteria isolated from blood cultures (9). It should be noted that the negative coagulase staphylococci are contaminants in the majority of cases and only 10% to 30% were clinically significant (10). Nevertheless, this distribution contrasts with data from a French study where S. aureus (18.4%), E. coli (15.4%) and S. epidermidis (14.4%) accounted for almost a half ( 48.3%) of blood cultures isolated microorganisms; Candida albicans represented 2.5%. A. baumannii was rarely isolated (0.6% of microorganisms responsible of nosocomial infections in all sites) (11).
Resistance to the 3 rd generation cephalosporins and ciprofloxacin, in Algeria, Tunisia and France, is over than 50% (11)(12)(13)(14) as it was in our study.
Our study objectified an increased resistance from 50% in 2010 to 86% in 2013 with a slight decrease in 2014 (75%). This could be due to pressure selection exerted by excessive prescription of imipenem.
The acquired carbapenem resistance in A. baumannii is often associated with carbapenemase production; IMP, VIM and SIM-type metallo-β-lactamase production or the OXA-24, OXA-23 and OXA-58 type class D carbapenemases. But also with the over production of natural oxacillinase (OXA-51) (15).OXA-23 and OXA-51 are considered as the most prevalent among A. baumannii carbapenem resistance (16). In our study, all A. baumannii strains were positive for bla OXA-51 genes and 87.5% (n=147) for bla  .
Strains of A. baumannii harbouring OXA-23 enzymes have been identified in Brazil, Argentina and Colombia (17)(18)(19)(20)(21). Moreover, carbapenemases belonging to OXA-23 subgroup have been detected in Europe, Australia, Tahiti, China, Korea, Singapore, Vietnam, USA, Libya and Pakistan (22) Mobilization of the bla OXA genes is determined by the presence of insertion sequences and transposons, and therefore has a high potential to spread (23). That is why it is essential to conduct molecular genotyping studies as well as characterizing the carbapenemases found in specific geographic areas, to highlight molecular evolution of both genes and resistant clones.
The multi-drug resistance of this germ has led to a renewed interest in colistin, to the association of antibiotics (rifampicin-colistin, colistin-imipenem) and the use of new antibiotics such as tigecycline to overcome therapeutic impasses (24).

CONCLUSION
A. baumannii was the second germ that was frequently isolated in blood cultures of intensive care units in the last five years in Ibn Rochd University Hospital. It was multi-resistant to antibiotics with a particular increasing resistance to imipenem. The strengthening of hospital hygiene measures and antibiotic resistance surveillance is needed to limit the spread of multi resistant bacteria and to optimize the management of antibiotic therapy.